Antibiotic resistance has increased dramatically in recent years and it is important to develop new strategies to treat bacterial infections. We propose a set of experiments to shed light on the molecular mechanisms by which two unusual substrate binding antibiotics, ramoplanin and nisin, kill bacterial cells. These molecules have very different structures, but both apparently target Lipid II, the disaccharide subunit of peptidoglycan. A better understanding of how these antibiotics function could lead to the ability to design improved antibiotics. The aims of the grant are summarized below. A) To define the structural requirements for substrate recognition by ramoplanin and nisin using synthetic Lipid I/Il substrate analogues. This work will provide crucial information about the selectivity of these molecules for carbohydrate moieties found on bacterial cell surfaces. B) To develop selective chemistry to modify ramoplanin; and to evaluate modified ramoplanin analogues for biological activity, Lipid ii binding, and ability to form fibrils upon binding to Lipid II. This work will shed light on the mechanism of ramoplanin; furthermore, information about where ramoplanin can be modified without interfering with activity will be useful for the design of other experiments to probe mechanism. C) To determine the structure of ramoplanin in non-aqueous solvents. This work may shed light on the bioactive conformation of ramoplanin and/or on how ramoplanin molecules self-associate when they bind Lipid H. D) To develop a synthetic approach to the synthesis of the N-terminal half of nisin and to evaluate nisin fragments for binding to Lipid II analogues. This work will shed light on the relationship between nisin structure and binding activity. It will also lay the groundwork for the design of hybrid antibiotics. E) To explore the feasibility of designing hybrid antibiotics targeted to bacterial cells by attaching the Lipid II binding domain of nisin to the pore forming antibiotic magainin.